The present invention relates to the first molecular genetic evidence, through the use of RFLP analysis, that an allele in the human dopamine (D.sub.2) receptor gene is more significantly associated with human brain tissue from alcoholics than with brain tissue obtained from nonalcoholics. The occurrence of this disease-associated polymorphism has a high predictive value in the classification of, at least, one probable subtype of alcoholics.
The identification of a genetic marker that is closely linked to alcoholism means that the gene's inheritance can be followed, leading to simple tests for diagnosing carriers and future disease victims, and potential gene therapy.
The tendency of certain individuals to display compulsive behavior patterns is well known and includes individuals with an excessive desire for substances classed as psychoactive drugs including, but not limited to alcohol, opiates, and food. Whether alcoholism is a psychiatric illness or a biological disease has been a controversial question, but there is some agreement that there are probably similar biochemical mechanisms for alcohol and opiates in terms of behavioral and pharmacological activities. (1)
Some authors believe that dopaminergic cells are implicated in the rewarding action of alcohol (23) and of opiates (2). In contrast, others (3) argue that at least alcohols/opiates and alcohol reinforcing effects are mediated primarily by nonadrenergic and not dopaminergic systems in the brain. Whether or not multiple systems exist, the question of several parallel reward mechanisms, or a very few, even one, is yet to be fully resolved. The cause and effect of compulsive behavior diseases, including alcoholism, appears to be biogenic. Regardless of the number of systems involved, the ability to identify an allelic gene segment associated with specific compulsive behavior is a significant step forward in developing predictive tests for compulsive behavior patterns.
Alcoholism is a major and devastating health problem with an unknown etiological basis. The question of whether environment or heredity is the prime determinant for the development of alcoholism continues to receive extensive attention throughout the world, and has recently involved the Supreme Court of the United States (4). However, family, twin, and adoption studies (5) are pointing to genetic factors as significant contributors to alcoholism. These studies also demonstrate that other forms of mental illness such as schizophrenia and other major psychoses are not found at higher frequencies in families of alcoholics compared with the general population. This would suggest that alcoholism is a primary disease.
Alcoholism currently is viewed as a heterogeneous entity arising from a combination of biopsychosocial factors (6). In regard to biological factors, an extensive literature reveals a wide range of potential physiological (7) and biochemical (8) markers in the risk for alcoholism. Moreover, family pedigree linkage analysis has implicated chromosomes 4, 6 and 11, but not specific gene markers, in the genetic risk for alcoholism (9).
Restriction Fragment Length Polymorphism (RFLP) offers a powerful molecular genetic tool for the direct analysis of the human genome to determine elements that provide predisposition to genetic diseases (10). This technique has been used to demonstrate a structural mutation in the gene that codes for an enzyme involved in alcohol metabolism (aldehyde dehydrogenase) which leads to the loss of this enzyme's ability to metabolize acetaldehyde. This altered gene is prevalent among Orientals (11) and may explain the well-known alcohol-flush syndrome as a protective factor in this population. However, no specific gene abnormality has been identified thus far which could regulate alcohol-seeking behavior, or is associated with alcoholism in humans.
Numerous studies indicate that, in animals, genetic control of neurotransmitter synthesis, metabolism, regulation, and receptor activity mediates reward in the meso-limbic circuitry of the brain (12), as well as drug (e.g., ethanol) - seeking behavior (13). In this regard, the dopamine.sub.2 (D.sub.2) receptor has been implicated as a prime target site in the N. accumbens and hippocampal CA.sub.1 cluster cells of the brain reward system (14). Three major dopaminergic systems in the human brain have been identified. The nigrostriatal is involved in the initiation and execution of movement; the tuberoinfundibular is responsible for the regulation of peptide secretion from the pituitary; and the mesolimbic tract controls emotional stability and affect. Mediating these effects of dopamine are two receptor subtypes, D.sub.1 and D.sub.2, each of which is coupled to different second messenger systems. The D.sub.1 receptor has been implicated in the sleep disorder, insomnia.
Important clinically relevant studies on the pharmacology of D.sub.2 receptors indicated that antipsychotic drugs display high affinities for the receptor. Other work suggested that the D.sub.2 receptor is involved in movement disorders, i.e., Parkinson's disease and tardive dyskinesia, tumors of the pituitary, and compulsive disease.
A cDNA encoding for rat dopamine (D.sub.2) receptor has been isolated (15). This receptor has been implicated in the pathophysiology of certain diseases, including drug addiction. The same laboratory localized the receptor gene to chromsome 11 (19). Partial sequence analysis revealed that the genomic clone lambda-hD.sub.2 G1 (ATCC #61354 and 61355) contains the last coding exon of the D.sub.2 receptor and 16.5 kb of 3-prime flanking sequence. When this clone was hybridized to human metaphase chromosomes and DNA from rodent-human hybrid cells, the data were consistent with a single human dopamine D.sub.2 receptor gene which mapped to the q22-q23 region of chromosome 11. This previous work provides a research tool to begin a molecular analysis of the human D.sub.2 receptor in alcoholism.
Access to sequence variation in the human genome now allows construction of genetic linkage maps through the technique of RFLPs (restriction fragment length polymorphisms). This technique provides probes which are isolated from chromosome specific phage libraries constructed to contain some portion of human DNA (16). With this tool in hand, the analysis of human gene segments is possible. The identification of an apparent gene abnormality in the tissue of alcoholics is an important advance in the art and of potential value in objectively identifying individuals who are genetically predisposed to alcoholism. The need for differential diagnosis and the ability to identify genetic predisposition has been recognized at the national level (17).
In the present invention, the DNA probe lambda-hD.sub.2 G1 (ATCC #61354 and 61355) effectively visualizes the human dopamine (D.sub.2) receptor gene. This permits evaluation of polymorphisms on the gene in a region close to the gene which could modify the function of the gene as a valuable predictor of alcoholism or other compulsive disorders.